The present invention relates to a method of plant cell culture. In particular, the present invention relates to a method for reducing browning of a plant cell culture.
The phenomenon of ‘browning’ is a significant problem encountered in the in vitro culture of plant cells. Browning (or sometimes ‘blackening’) of cultured plant material occurs when phenolic substances, such as tannins or other hydroxyphenols, are oxidised to highly active quinone compounds which then cyclise, polymerise and/or oxidise proteins to form increasingly melanic compounds. The result of this is that the plant tissue becomes brown or black and growth of the plant cells in culture is inhibited, frequently irreparably. Thus, browning represents a serious obstacle to the successful growth of plant cells in culture.
Browning has been observed in a wide range of plant explants and plant cell cultures and in particular in tropical species, which often contain high levels of phenolic substances.
A number of approaches have been taken in attempting to address the problem of browning. These include the addition of anti-oxidants, such as ascorbic acid, and chelating agents, such a EDTA, and the use of activated charcoal to absorb inhibitory phenolic compounds. However, no single approach has proved satisfactory, and it has frequently been necessary to combine several of these techniques. A drawback of the use of activated charcoal is that as well as absorbing problematic phenolic compounds, it may also absorb growth factors or other mediators added to the culture medium and therefore higher amounts of the growth factors or other mediators must be added. Even in combination, the previously adopted techniques have often failed to control browning in plant cell cultures.
One plant known to suffer from browning in culture is the commercially important crop plant, sugar cane. Sugar cane is a perennial tropical grass with tall unbranched stems or canes from which sugar is extracted. The sugar cane stem is roughly circular or oval in cross-section, and comprises a series of joints each of which in turn comprises a node and an internode. Each node comprises of a lateral bud in an axial leaf, a band containing root primordia, and a growth ring.
In commercial production, sugar cane is usually grown from stem cuttings (or ‘setts’), each of which generally has at least two buds. The buds develop to form primary stems, the basal buds of which in turn develop into secondary stems, from which tertiary stems may develop, and so on.
Sugar cane roots may develop either from the root primordia of a cutting itself or from the root primordia of stem shoots developing from the cutting, each of which produce their own root system. Roots developing from the primordia of the sugar cane stem cutting tend to be thin and branched. Those formed by the root primordia of new stem shoots tend to be thicker and less branched. Sugar cane stem cuttings may be rooted in water or an aqueous medium. It has been reported that sugar cane roots can develop at temperatures as low as around 10° C.